Urinary Copper Elevation in a Mouse Model of Wilson's Disease Is a Regulated Process to Specifically Decrease the Hepatic Copper Load

Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA.
PLoS ONE (Impact Factor: 3.23). 06/2012; 7(6):e38327. DOI: 10.1371/journal.pone.0038327
Source: PubMed


Body copper homeostasis is regulated by the liver, which removes excess copper via bile. In Wilson's disease (WD), this function is disrupted due to inactivation of the copper transporter ATP7B resulting in hepatic copper overload. High urinary copper is a diagnostic feature of WD linked to liver malfunction; the mechanism behind urinary copper elevation is not fully understood. Using Positron Emission Tomography-Computed Tomography (PET-CT) imaging of live Atp7b(-/-) mice at different stages of disease, a longitudinal metal analysis, and characterization of copper-binding molecules, we show that urinary copper elevation is a specific regulatory process mediated by distinct molecules. PET-CT and atomic absorption spectroscopy directly demonstrate an age-dependent decrease in the capacity of Atp7b(-/-) livers to accumulate copper, concomitant with an increase in urinary copper. This reciprocal relationship is specific for copper, indicating that cell necrosis is not the primary cause for the initial phase of metal elevation in the urine. Instead, the urinary copper increase is associated with the down-regulation of the copper-transporter Ctr1 in the liver and appearance of a 2 kDa Small Copper Carrier, SCC, in the urine. SCC is also elevated in the urine of the liver-specific Ctr1(-/-) knockouts, which have normal ATP7B function, suggesting that SCC is a normal metabolite carrying copper in the serum. In agreement with this hypothesis, partially purified SCC-Cu competes with free copper for uptake by Ctr1. Thus, hepatic down-regulation of Ctr1 allows switching to an SCC-mediated removal of copper via kidney when liver function is impaired. These results demonstrate that the body regulates copper export through more than one mechanism; better understanding of urinary copper excretion may contribute to an improved diagnosis and monitoring of WD.

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    • "As suggested in previous studies [18], [28], [29], copper is supposed to progressively accumulate in LEC hepatocytes during the first weeks of life until cytosolic storage capacities are overwhelmed. This could be accelerated by a massive burden of dietary copper as recently reported by Siaj et al. [18]. "
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    ABSTRACT: Wilson's disease (WD) is an inherited disorder of copper metabolism leading to liver failure and/or neurological impairment. Its diagnosis often remains difficult even with genetic testing. Relative exchangeable copper (REC) has recently been described as a reliable serum diagnostic marker for WD. The aim of this study was to validate the use of REC in the Long Evans Cinnamon (LEC) rat, an animal model for WD, and to study its relevance under different conditions in comparison with conventional markers. Two groups of LEC rats and one group of Long-Evans (LE) rats were clinically and biologically monitored from 6 to 28 weeks of age. One group of LEC rats was given copper-free food. The other groups had normal food. Blood samples were collected each month and different serum markers for WD (namely ceruloplasmin oxidase activity, exchangeable copper (CuEXC), total serum copper and REC) and acute liver failure (serum transaminases and bilirubinemia) were tested. Every LEC rat under normal food developed acute liver failure (ALF), with 40% global mortality. Serum transaminases and bilirubinemia along with total serum copper and exchangeable copper levels increased with the onset of acute liver failure. A correlation was observed between CuEXC values and the severity of ALF. Cut-off values were different between young and adult rats and evolved because of age and/or liver failure. Only REC, with values >19%, was able to discriminate LEC groups from the LE control group at every time point in the study. REC sensitivity and specificity reached 100% in adults rats. REC appears to be independent of demographic or clinical data in LEC rats. It is a very simple and reliable blood test for the diagnosis of copper toxicosis owing to a lack of ATP7B function. CuEXC can be used as an accurate biomarker of copper overload.
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    • "n , the regulation of intracellular Cu is complex and other yet to be characterized Cu transport path - ways may also be altered in PD . It has recently been shown , for example , that downregulation of Ctr1 in a murine model of Wilson disease is linked to the appearance of a redundant mechanism of Cu transport , the 2 - kDa small copper carrier ( Gray et al . , 2012 ) . A second Ctr protein , structurally related to Ctr1 , has also been identified in mammals , designated Copper transporter 2 ( Ctr2 ) . This protein has not been identified in human tissue but is thought to play a role in low - affinity Cu import ( Bertinato et al . , 2008 ) and in intracellular Cu mobilization ( Rees and Thiele , 20"
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    • "The decreased transcription activity of CTR1 and the low CTR1 protein production in WND patients that we have documented may be a plausible adaptative mechanism, which aims to reduce copper influx into enterocytes. Our results seem to be in line with previous observations in mice with selective Atp7b gene knockout in the liver (an animal model of WND) (Gray et al. 2012). These mice presented low Ctr1 expression in hepatocytes as compared to wild littermates . "
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    ABSTRACT: In Wilson's disease (WND), biallelic ATP7B gene mutation is responsible for pathological copper accumulation in the liver, brain and other organs. It has been proposed that copper transporter 1 (CTR1) and the divalent metal transporter 1 (DMT1) translocate copper across the human intestinal epithelium, while Cu-ATPases: ATP7A and ATP7B serve as copper efflux pumps. In this study, we investigated the expression of CTR1, DMT1 and ATP7A in the intestines of both WND patients and healthy controls to examine whether any adaptive mechanisms to systemic copper overload function in the enterocytes. Duodenal biopsy samples were taken from 108 patients with Wilson's disease and from 90 controls. CTR1, DMT1, ATP7A and ATP7B expression was assessed by polymerase chain reaction and Western blot. Duodenal CTR1 mRNA and protein expression was decreased in WND patients in comparison to control subjects, while ATP7A mRNA and protein production was increased. The variable expression of copper transporters may serve as a defense mechanism against systemic copper overload resulting from functional impairment of ATP7B.
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